The major objectives of the project are the elucidation of the mechanisms by which insulin, catecholamines, thyroid hormones, growth hormone and other agents regulate the metabolism of isolated rat and rabbit fat cells. Emphasis is placed on the regulation of lipid metabolism in adipose tissue. The specific areas being investigated are: 1. The relationship of cyclic AMP accumulation to lipolysis and the regulation of adenylate cyclase by catecholamines, thyroid hormones, glucocorticoids, and growth hormone. Pertussis toxin will be used as a tool to look at the role of the inhibitory guanine nucleotide binding protein (Ni) in the action of hormones. We are especially interested in the mechanisms by which pertussis toxin restores cyclic AMP responsiveness of adipocytes from hypothyroid rats to norepinephrine or forskolin without correcting the defect in lipolysis. Stimulation by catecholamines of triacylglycerol lipase phosphorylation will be correlated with cyclic AMP accumulation and lipolysis in adipocytes in response to catecholamines and forskolin in adipocytes from hypothyroid, euthyroid and hyperthyroid rats. 2. The relationship of phosphatidylinositol turnover to insulin and alpha 1 catecholamine agonist action in adipocytes and the mechanisms by which pertussis toxin blocks alpha1 adrenergic activation of phosphatidylinositol turnover will be investigated. A search will be made for conditions in which activation of phosphatidylinositol breakdown can be seen in adipocytes, especially that of phosphatidylinositol 4,5-bisphosphate during the first 30 sec after hormone addition. An attempt will also be made to demonstrate normonal activation of phosphoinositide breakdown in prelabeled adipocyte plasma membrane fractions. The studies are designed to provide basic information about diseases with disordered lipid metabolism such as diabetes and atherosclerosis.